The goal of this proposal is the elucidation of the genetic pathway underlying embryonic hematopoiesis in a vertebrate species. We choose to study the regulation of GATA-1, one of the earlist expressed genes required for erythroid development, as an entry point to decipher this pathway. Presently, little is known regarding factors that function early to initiate GATA-1 expression or that cooperate with GATA-1 to establish the erythroid lineage during vertebrate embryogenesis. The use of an in vivo system that allows one to integrate approaches of experimental embryology, genetics and molecular biology would greatly facilitate identification of these factors. We seek to identify these factors in the zebrafish, were chemical mutagenesis screens recently have led to the isolation of several mutants defective in embryonic blood cell formation. By injecting DNA constructs containing the GATA-1 promoter ligated to the green fluorescent protein (GFP) reporter gene, we have generated GATA-1-GFP transgenic fish that faithfully recapitulate the GATA-1 expression pattern in living embryos. These fish make it possible to observe continuously the dynamic expression patterns of GATA-1 in vivo and to isolate GATA-1 expressing cells for in vitro studies. Using this system, coupled to genetic analysis of embryonic blood mutations, we have developed approaches that allow rapid identification of novel genes that are expressed specifically in hematopoietic progenitor cells during embryongenesis. We propose to characterize the novel hematopoietic genes that we have already identified and to identify additional candidates. Their potential roles in hematopoietic development will be examined in wild type embryos and in embryos with defects in blood development. Given the fact that the expression pattern and sequences of many important hematopoietic genes are well conserved between fish, mice and humans, our studies should ultimately lead to a better understanding of the molecular and genetic bases underlying initial specification of mammalian hematopoietic progenitor cells.